Genetics of the formation of repair & recombination foci

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DESCRIPTION (provided by applicant): DMA rearrangements occur in response to many genomic insults and are often associated with neoplasia as well as with other disease states (e.g., immuno-deficiencies, thalassemias etc.). In cells ranging from simple organisms such as yeast and bacteria up to humans, recombination and repair proteins localize to distinct foci in the nucleus in response to DNA damage. These foci also form spontaneously during DNA synthesis and segregation. To examine the genetic control of these processes, we will look at the formation and disassembly of repair/recombination foci using Saccharomyces cerevisiae as a model system. We will examine these processes in living yeast cells using time-lapse microscopy of multi-labeled cellular components. We will engineer reagents that will allow us to dissect the genetic pathways responsible for focus formation. As this is likely a coordinated process with the cell cycle, we will pay particular attention to cell cycle checkpoint proteins for their effects on focus formation. We have already developed methods that will aid in our studies and we propose to develop new reagents that will permit a further glimpse into the in vivo action of key components of this system. Our specific aims are: (1) To molecularly and genetically dissect checkpoint and repair foci using CFP, YFP and DsRed to specifically label chromosomal DSB sites and repair proteins. (2) To address the coordination of DNA replication, repair and checkpoint activation, which stems from our observation that repair of DSBs specifically occurs in S phase (or G2) of the cell cycle. Chemical agents as well as genome instability mutants will be examined. (3) To explore the dynamics of focus assembly/disassembly by using time-lapse microscopy, fluorescent recovery after photobleaching (FRAP) and fluorescent loss in photobleaching (FLIP) analyses. (4) to develop a new approach to visualize the timing of important biological events in vivo by fusing DsRed to antibodies that specifically recognize DNA damage-induced epitopes of central repair and recombination proteins. These approaches are general and the insights that we gain and the methods that we develop will not be confined to yeast alone, but will be applicable to many cellular systems.